purpose. To assess the distribution of transglutaminase (TGase) activity in
ocular tissues and the target structures for cross-linking.

methods. Cryosections from human and cynomolgus monkey eyes were incubated with
the biotinylated amine donor substrate cadaverine (biotC), which was
subsequently visualized with streptavidin-peroxidase. Confocal laser
scanning was used to colocalize biotC and fibrillin, a major component
of elastic microfibrils and the zonular fibers in particular.
Cryosections and isolated bovine zonules were treated with purified
TGase 2 and biotC. The distribution of different TGases (1, 2, 3, and
factor XIII) was confirmed immunohistochemically.

results. Virtually all ocular tissues showed TGase activity with a remarkable
preponderance for the ciliary body, zonular fibers, and blood vessel
walls. Confocal laser scanning revealed fibrillin-containing
microfibrils as a major target for TGase activity, in particular the
ciliary zonules. Corneal epithelium and basement membrane showed a
TGase cross-linking pattern similar to skin. Treatment of cryosections
and isolated bovine zonular fibers with purified TGase 2 led to
additional incorporation of biotC into extracellular matrix,
particularly zonular fibers. The immunohistochemically predominant
TGase 2 was associated with epithelia and particularly with connective
tissue fibers. TGase 1 was restricted to the corneal epithelium,
whereas factor XIII was found to be associated only with blood vessels.
TGase 3 was absent.

conclusions. TGase 2 appears to be an important cross-linker and thus stabilizer of
ocular connective tissue. In particular, the zonular fibers are a major
target for TGase 2. This is of relevance in hereditary
microfibrillopathies such as Marfan syndrome, which exhibits distinct
ocular manifestations such as elongated bulbus, retinal detachment, and
subluxation of the lens. Purified or recombinant TGase might be of
therapeutic use in the future.

Transglutaminases (TGases; EC 2.3.2.13) form a family of enzymes
that stabilizes protein assemblies by γ-glutamyl-ε-lysine
cross-links.12 Seven different genes are currently known
in higher vertebrates.34 Obviously, the individual TGases
have different substrate specificities—that is, factor XIIIa
stabilizes the fibrin clot in hemostasis, whereas TGases 1 and 3
cross-link different intracellular proteins of the cornified envelope
in differentiating epidermis.3 Accordingly, congenital
deficiency of factor XIII causes reduced blood clot
stability,5 whereas mutations in the TGase 1 gene underlie
one form of autosomal recessive lamellar ichthyosis.67 Less is understood concerning the physiological function of tissue-type
TGase (TGase 2), which is expressed widely in
vertebrates.389 Besides a role in guanosine triphosphate
(GTP)–binding in receptor signaling,10 and
apoptosis,11 cross-linking of extracellular matrix
proteins has been proposed.312 In fact,γ
-glutamyl-ε-lysine cross-links have recently been demonstrated in
osteonectin,912 fibronectin,13 and
heterotypic collagen V/XI fibrils.14 Recently, anchoring
fibrils of the basement membrane zones of skin and the cornea and their
major component, collagen VII, were identified as substrate for TGase
2.15 Because this points to an important role of TGase 2
in the stabilization of tissue and maintenance of
epithelial–mesenchymal cohesion, we studied the distribution of TGase
activity in the human and cynomolgus monkey eye.

Materials and Methods

TGase Substrates and Inhibitors

A 10-mM stock solution of
biotinyl-5-(N-biotinoyl-amino-hexanoyl-pentylamine)
(biotin-X-cadaverine, or biotC; Molecular Probes Europe, Leiden, The
Netherlands) was freshly prepared by dissolving 7 mg in 300 μl 0.1 M
HCl and subsequent addition of 1700 μl distilled water. A 100-mM
stock solution of putrescine (diaminobutane; Fluka, Buchs, Switzerland)
was made in water. A 200-mM stock solution of EDTA was made in water
and the pH adjusted to 7.5 with sodium hydroxide.

Visualization of Endogenous TGase Activity in Ocular Tissues

The eyes of a female cynomolgus monkey (Macaca
fascicularis) were snap frozen in toto in melting isopentane.
Cryosections were examined of anterior and posterior segment tissue
from five normal human eyes (age range, 46–82 years) obtained at
autopsy and snap frozen in liquid nitrogen-isopentane 2.5 to 8 hours
after death. The eyes had no history or morphologic evidence of ocular
disease. Ten- to 12-μm cryostat sections of whole eyes (cynomolgus
monkey) or 7-μm cryosections of pretrimmed tissue blocks (human eyes)
were incubated with 1% bovine serum albumin in 0.1 M Tris/HCl (pH 8.2)
for 30 minutes at room temperature to block nonspecific binding. TGase
activity was detected by subsequent incubation in the same buffer
containing 100 μM biotC and 10 mM
CaCl2.916 Control sections were
incubated with the same biotC-supplemented buffer containing in
additional 2 mM putrescine or 10 mM EDTA, instead of
CaCl2. The enzyme reaction was allowed to proceed
for 2 hours at room temperature, was stopped by washing the slides for
5 minutes in phosphate-buffered saline (PBS) containing 10 mM EDTA, and
was followed by two further washings in plain PBS. Light microscopic
visualization of transamidated ocular structures was performed with
streptavidin-peroxidase (Jackson Immunoresearch, West Grove, PA), and
the chromogenic reaction was performed with aminoethylcarbazol
with a kit from Dako (Glostrup, Denmark). In this case sections were
lightly counterstained with hemalum and embedded in gelatin. For double
immunofluorescence the polyclonal antibody PF2 against the peptic
fragment 2 of fibrillin (1:100 in PBS) was used. BiotC was visualized
using dichlorotriazinaminofluorescein-conjugated streptavidin (1:100;
Jackson Immunoresearch), the fibrillin antibody with a Texas
red–coupled goat anti-rabbit IgG (1:100; Jackson Immunoresearch).
Preparations were mounted in Mowiol (Hoechst, Frankfurt am Main,
Germany) in Tris/HCl (pH 8.6) and examined using an inverted
confocal laser scanning microscope (LSM 410; Carl Zeiss, Oberkochen,
Germany) combined with two HeNe lasers (543 and 633 nm) and an argon
laser (488 nm) for multicolor fluorescence.

Immunogold Electron Microscopy of Isolated Bovine Zonular Fibrils

Bovine eyes were obtained from animals 6 to 8 months old
(PelFreez, Rogers, AR) and shipped overnight on ice. Zonules were
dissected and washed in 0.01 M PBS plus protease inhibitors (0.002 M
EDTA, 0.01 M N-ethylmaleimide, and 0.001 M
phenylmethylsulfonyl fluoride). One milligram zonular fibrils was
sedimented at 14,000g for 30 minutes and redispersed in
500 μl 100 mM Tris buffer (pH 8.3) containing 10 mM
CaCl2, 200 μM biotC, and 12 μg purified guinea pig
liver TGase 2 and incubated overnight at 25°C. Eye cryosections were
treated under the same conditions. In control experiments TGase was
omitted or the enzymatic activity inhibited by the addition of 50 mM
EDTA. Zonular fibrils were washed three times in PBS, and aliquots were
adsorbed for 2 minutes onto Formvar (Sigma, St. Louis, MO)
carbon-coated copper grids. The grids were washed with PBS and were
treated for 30 minutes with 2% (wt/vol) dried skim milk in PBS. The
adsorbed material was then allowed to react for 2 hours at room
temperature with rabbit anti-fibrillin antibody PF2 (1:50 in 0.2%
wt/vol) dried skim milk and PBS. After the grids were washed five times
for 2 minutes with PBS, they were incubated for 2 hours with a
suspension of 6 nm colloidal gold particles coated with streptavidin
(Amersham Buchler, Braunschweig, Germany) and 12 nm colloidal gold
particles coated with IgG against rabbit immunoglobulins (Dianova,
Hamburg, Germany). Finally, the grids were washed with PBS and
negatively stained with 2% uranyl acetate. Micrographs were taken at
80 kV with an electron microscope (CM 10; Philips, Einthoven, The
Netherlands). Cryosections were processed as has been
described.

In the cornea, biotC incorporation was detected in the epithelium,
predominantly within the intercellular spaces but also in the cytoplasm
of the epithelial cells and along their basement membranes. Keratocytes
in the superficial layers of the corneal stroma were also labeled (Fig. 1A ). TGase activity was visualized in the conjunctival epithelium,
especially within intercellular spaces (Fig. 1B) . Further cross-linking
of biotC was detected in the walls of stromal vessels. In the sclera
the presence of TGase activity was restricted to the endothelial lining
of intra- and episcleral blood vessels (Fig. 1C) and single scattered
cells, presumably fibroblasts, between the collagen lamellae. In the
trabecular meshwork TGase activity was found in association with the
trabecular endothelial cells, particularly in the posterior part of the
meshwork (Fig. 2A ) and in association with the endothelial cells lining Schlemm’s
canal, collector channels, and intrascleral aqueous veins. In the iris
BiotC incorporation was prominent in the smooth muscle cells of the
dilator and sphincter muscles, in the vascular endothelial cells of
stromal vessels, and along delicate fibrillar structures in the stroma,
which were particularly concentrated in the anterior boarder layer (Fig. 1D) . Extensive cross-linking was apparent along most ciliary body
structures, especially along the zonular fibers covering the surface of
the ciliary epithelium, and also in the ciliary muscle cells and the
outer limiting membrane (i.e., the basement membrane of the pigmented
ciliary epithelium; Figs. 2A2B2C2D ). The inner limiting membrane
(the basement membrane of the nonpigmented epithelial layer) expressed
some TGase activity in the pars plana region only. The nonpigmented
epithelial layer was essentially negative in the pars plana area. The
stromal connective tissue of the ciliary body was characterized by
TGase activity in stromal cells (presumably fibrocytes), in vascular
endothelial cells, and along extracellular fibrillar strands. In the
lens biotC incorporation was strong in the zonula lamella on the
surfaces of the lens capsule and in the zonular fibers adhering to it
and to a minor extent in the lens epithelial cells. Some TGase activity
was also detected in the equatorial portions of the lens capsule (not
shown). The choroid expressed high-level TGase activity within cells
and fibrous strands of the connective tissue, within Bruch’s membrane,
and within vascular endothelial cells of the choriocapillaris (Fig. 3A ). TGase activity in the retina was strictly confined to the
endothelial lining of retinal capillaries (Fig. 3A) . Activity in the
optic nerve was localized to capillaries within the glial columns of
the prelaminar portion, to the cribriform plates of connective tissue
in the laminar portion, and to the connective tissue septa including
their vasculature in the postlaminar portion (Fig. 3C) . Additional
cross-linking was found in the optic nerve meninges, particularly the
pia mater and arachnoidea (not shown). EDTA completely inhibited biotC
incorporation, demonstrating the specificity of the reaction (Figs. 2B , 3B , and 3D ).

Monkey Eye.

BiotC incorporation into whole monkey eye sections highly resembled the
pattern obtained for human eyes. Endogenous TGase activity was detected
in the corneal epithelium and its basement membrane and in superficial
stromal keratocytes, in the trabecular endothelial cells in low
amounts, and in the dilator and sphincter muscles of the iris and
vessel walls of the iris stroma. Extensive TGase cross-linking sites
were again observed in the ciliary body (Figs. 4A , 4B ), particularly in the zonular fibers on the surface, in the
ciliary muscle, the outer limiting membrane, the inner limiting
membrane in the pars plana region, and the stromal connective tissue
including vessel walls. The nonpigmented ciliary epithelium was only
weakly labeled in the region of the pars plicata. BiotC incorporation
was further localized to the lens epithelium, the zonular lamella on
the surface of the lens capsule, and equatorial portions of the lens
capsule proper. TGase activity was also apparent in retinal capillaries
(Figs. 4C , 4D ), in stroma and vasculature of the choroid, and in
episcleral vessels. In addition, biotC incorporation was prominent in
the sclera in association with fibrillar structures (Fig. 4D) . In the
conjunctiva, biotC cross-linking was found in the conjunctival
epithelium, in its basement membrane, and in association with vessel
walls and fibrillar structures in the stroma, which appeared to be
particularly concentrated subjacent to the epithelial layer (Figs. 4E , 4F ). A characteristic pattern of cross-linking surrounded all
individual muscle fibers of extraocular muscle tissue, which was not
included in the human specimens (Fig. 4D) . The optic nerve was not
included in the monkey eye sections.

TGase Activity in Fibrillin-Containing Microfibrils

The observed distribution of fibrillin, the major component of 10-
to 12-nm microfibrils was highly comparable in both species and was in
very good accordance with earlier findings in human
eyes.1819 In particular, the epithelial basement membrane
region consistently exhibited fibrillin staining, which was most
prominent in the peripheral cornea (Fig. 5A ) but faint in the central cornea (not shown). The sclera showed
interlamellar fibrillin staining, predominantly in the anterior part (Fig. 5b) . In the retina, fibrillin was present only in the walls of
retinal vessels (Fig. 5C) . Fibrillin in the optic nerve was localized
to capillaries within the glial columns of the prelaminar portion, to
the cribriform plates of connective tissue in the laminar portion, and
to the connective tissue septa including their vasculature in the
postlaminar portion (Fig. 5D) . Fibrillin was also present in the optic
nerve meninges, particularly the pia mater and arachnoidea (not shown).
A particularly strong signal for fibrillin was obtained with ciliary
zonules (Fig. 5E) , a moderate signal in the adventitia of conjunctival
blood vessels (Fig. 5F) and endomysial sheets in eye muscle (data
obtained only in monkey eyes; not shown). TGase activity showed a
broader distribution than that of fibrillin; however, there was
extensive colocalization of fibrillin and TGase activity, which became
evident after superimposition of images (Fig. 5) .

Transamidation of Isolated Zonules with Purified TGase 2

In the presence of exogenous TGase 2, the pattern of biotC
incorporation was not significantly altered, except for an increased
reaction in the iris stroma (both cell-bound and along thin, delicate
fibrillar structures), in the trabecular meshwork (cell-bound), in the
equatorial portions of the lens capsule, and in the photoreceptor layer
(inner segments) of the retina. This demonstrates that these contained
substrate sites for the enzyme that were not cross-linked under normal
conditions (data not shown). The zonular fibers again appeared as a
prominent target structure, which led us to treat isolated bovine
zonules in suspension with purified guinea pig liver TGase 2. This led
to incorporation of biotC, which was not evenly distributed along the
entire zonules but largely clustered, probably because of masked or
saturated cross-linking sites (Fig. 6) .

Distribution of Various TGases in the Eye: Predominance of TGase 2

Immunhistochemically, we could demonstrate TGase 2 in comparison
with TGases 1 and 3 and factor XIII as the predominant enzyme in ocular
tissue. TGase 2 was associated extracellularly with fibers in the
stromata of the ciliary body (Fig. 7A ), iris (Fig. 7B) , and conjunctiva (not shown). Cell-associated TGase 2
was noted within corneal epithelium (not shown), endothelia, and single
cells in the iris stroma (Fig. 7A , 7B ). There was also moderate
staining of endomysial sheets of rectus muscle (not shown). The ciliary
zonules were negative in human eyes and only faintly positive in the
cynomolgus monkey eye (not shown). The other TGases were not found in
association with connective tissue structures. TGase 1 was identified
exclusively in the corneal epithelium, mostly in suprabasal cells (Fig. 8A ). Factor XIII was associated with the endothelium of arterioles in the
conjunctival stroma (Fig. 8B) and capillaries (Fig. 8C) . TGase 3 was
not absent in the eye but was visualized in human skin as a positive
control in the cytoplasm of the granular layer (not shown).

Discussion

With the histochemical methods used here, we demonstrated
substantial TGase activity in various ocular tissues in situ. There is
only limited information in the literature on the expression pattern of
TGases in different tissues of the eye, and that prompted us to conduct
this study. Prior research focused primarily on the lens, because it
had been suggested that TGase cross-linking may contribute to cataract
formation, based on the finding that TGase activity andγ
-glutamyl-ε-lysine cross-links are increased by an order of
magnitude in senile cataract both in humans and in animal
models.2021 β-Crystallins,212223 and more
recently, vimentin24 have been identified as the target
proteins for TGase cross-linking in lens. TGase 2 has been implicated
in this process20 and shown to be expressed by the
anterior lens epithelium,25 consistent with our results.
An induction of TGase 2 expression has also been associated with
programmed cell death in various cell types,11 and TGase 2
has recently been shown to be involved in apoptosis of retinal
photoreceptor cells after both in photic injury and in the rat model
for hereditary retinal dystrophy.26 Accordingly, we have
not seen significant levels of TGase activity in retinal neurons in the
normal eye.

There is also not much information available on the presence of other
TGases in the eye. Band 4.2, a TGaselike molecule without cross-linking
activity, has been detected in bovine and chicken eye lens and possibly
participates in the architecture of the lens fiber cell
membranes.27 TGase 1 was only recently investigated and
found by in situ hybridization to be expressed in conjunctival
epithelium of patients with Stevens–Johnson syndrome, but not in
normal conjunctival epithelium (data on the corneal epithelium are not
reported).28 Our immunohistochemical data also point to an
absence of TGase 1 in normal conjunctival epithelium; however, they
show presence of this protein in corneal epithelium and, in contrast to
skin, the absence of TGase 3. No antibodies are currently available to
identify the recently identified TGase X4 in tissue.

In particular, we have found the bulk of TGase activity to be
extensively associated with connective tissue structures, in particular
fibrillin-containing microfibrils. As a scaffold for elastin
deposition, microfibrils facilitate elastic fiber
formation29 in a large variety of
tissues2630 but also exist as elastin-free bundles in the
papillary layer of the human dermis and in the suspensory ligament of
the lens. Our findings extend recent direct chemical evidence forγ
-glutamyl-ε-lysine cross-links in trypsinized microfibrils
isolated from human amnion31 and in microfibrils derived
from invertebrates.32 As the major constituent of
microfibrils,33 fibrillin can potentially form
homopolymers stabilized by intermolecular cross-links. This has been
suggested by the characterization of one (of several possible)
fibrillin–fibrillin TGase-derived cross-link.31 As for
the immunohistochemical localization of TGases, we did not expect a
priori an identical distribution of protein and activity, because the
enzyme could be present but inactive at certain locations. However, we
could immunolocalize the TGase 2 protein in many areas of
histochemically detected TGase activity. As an exception, we could not
localize TGase 2 immunohistochemically to the zonule fibrils, which
otherwise displayed strong TGase activity and were an excellent target
for purified guinea pig TGase 2. This may have been because of the
autocatalytic activity of TGases. If the enzymes stays in place, it may
mask its own epitope for a monoclonal antibody.

However, other microfibrillar components may also be partners for
fibrillin in TGase cross-links. Not much is known presently about the
composition of microfibrils except for the microfibril-associated
glycoprotein (MAGP)-1, which has recently been localized to the beaded
domains of microfibrils from bovine zonules.34 Interestingly, MAGP has been shown to be a glutaminyl substrate for
TGase 2.35 Other microfibril-associated proteins are
latent transforming growth factor–binding proteins
(LTBP)-136 and LTBP-2.37 Apparently, LTBP-1
is also a substrate for TGase 238 but appears to be
immunohistochemically absent from zonular fibrils36 and
cyanogen bromide preparations of isolated zonule
fibrils.39

Microfibrillar defects due to fibrillin mutations play a major role in
Marfan syndrome40 and its ocular manifestations, such as
ectopia lentis, dehiscences of suspensory ligaments, myopia, retinal
detachment, presenile cataracts, glaucoma, iris abnormalities, and
corneal flattening.414243 Extensive ultrastructural
studies of microfibrils extracted from fibroblast cultures of Marfan
patients have consistently shown abnormalities.44 Remarkably, microfibrils formed in the presence of TGase inhibitors in
normal fibroblast cultures also show considerable structural
alterations (MR et al., unpublished data, July, 1996). It is
therefore tempting to speculate that fibrillin mutations disrupting or
deleting sites for TGase cross-linking can cause the microfibrillar
instability that results in the ocular abnormalities found in the
Marfan syndrome or related phenotypes. This may be particularly
interesting for the pseudoexfoliation syndrome, in which excessive
production and abnormal aggregation of fibrillin-containing
microfibrils have been proposed and demonstrated.4546 Finally, cataract research is currently focusing on inhibitors against
the TGase-catalyzed cross-linking of lens proteins.47 In
this regard our data underline that the area and place make TGase
activity beneficial or disease causing.

Supported by the German Science Foundation Grants Ra 447/3 (MR) and Ra 447/3 (RC) and by National Institutes of Health Grant EY09908.

Visualization of endogenous TGase activity in human ocular tissues
(A) Cornea: TGase activity was present in the corneal
epithelium, its basement membrane, and keratocytes of the superficial
stroma. (B) Conjunctiva: BiotC incorporation was prominent
in the conjunctival epithelium, particularly along the intercellular
spaces. (C) Sclera: BiotC was predominantly incorporated
into episcleral vessel walls. (D) Iris: TGase activity was
present in the dilator muscle, in the walls of stromal blood vessels,
and along delicate fibrillar structures in the stroma, particularly in
the anterior border layer. Bars, 100 μm.

Figure 1.

Visualization of endogenous TGase activity in human ocular tissues
(A) Cornea: TGase activity was present in the corneal
epithelium, its basement membrane, and keratocytes of the superficial
stroma. (B) Conjunctiva: BiotC incorporation was prominent
in the conjunctival epithelium, particularly along the intercellular
spaces. (C) Sclera: BiotC was predominantly incorporated
into episcleral vessel walls. (D) Iris: TGase activity was
present in the dilator muscle, in the walls of stromal blood vessels,
and along delicate fibrillar structures in the stroma, particularly in
the anterior border layer. Bars, 100 μm.

TGase activity in cynomolgus monkey eye. (A) Pars plicata
region of the ciliary body: BiotC was strongly incorporated into the
zonular fibers on the surface and moderately incorporated into the
nonpigmented ciliary epithelium, the connective tissue stroma, and the
ciliary muscle itself. (B) Pars plana region of the ciliary
body: TGase activity was visualized in the zonular fibers on the
surface of the nonreactive ciliary epithelium, in the inner limiting
membrane, the connective tissue layer, the extension of the ciliary
muscle, and the sclera. (C) Retina and choroid: In addition
to the choroidal connective tissue, biotC incorporation was confined to
the walls of the retinal capillaries and, to a minor extent, to the
inner segments of the photoreceptor layer. (D) Retina,
choroid, sclera, and extraocular muscle tissue: TGase activity was
demonstrated in retinal vessels walls, in the choroid and sclera, and
in the vessel walls and margins of individual muscle fibers of
extraocular muscle tissue. (E) Conjunctiva: BiotC was
incorporated into epithelial cells, walls of conjunctival and
episcleral vessels, and fine fibrillar structures in the conjunctival
stroma, particularly concentrating subjacent to the epithelial layer.
(F) Higher magnification of (E). Bars, 100μ
m.

Figure 4.

TGase activity in cynomolgus monkey eye. (A) Pars plicata
region of the ciliary body: BiotC was strongly incorporated into the
zonular fibers on the surface and moderately incorporated into the
nonpigmented ciliary epithelium, the connective tissue stroma, and the
ciliary muscle itself. (B) Pars plana region of the ciliary
body: TGase activity was visualized in the zonular fibers on the
surface of the nonreactive ciliary epithelium, in the inner limiting
membrane, the connective tissue layer, the extension of the ciliary
muscle, and the sclera. (C) Retina and choroid: In addition
to the choroidal connective tissue, biotC incorporation was confined to
the walls of the retinal capillaries and, to a minor extent, to the
inner segments of the photoreceptor layer. (D) Retina,
choroid, sclera, and extraocular muscle tissue: TGase activity was
demonstrated in retinal vessels walls, in the choroid and sclera, and
in the vessel walls and margins of individual muscle fibers of
extraocular muscle tissue. (E) Conjunctiva: BiotC was
incorporated into epithelial cells, walls of conjunctival and
episcleral vessels, and fine fibrillar structures in the conjunctival
stroma, particularly concentrating subjacent to the epithelial layer.
(F) Higher magnification of (E). Bars, 100μ
m.

Immunfluorescence detection of TGase 2 in human eye.
(A) Ciliary body TGase 2 was present in connective tissue
fibers of the ciliary stroma and endothelia and associated
extracellularly with fibers in the stromata of the ciliary body.
(B) In the iris the enzyme appeared to be associated with
endothelia and single cells in the iris stroma. Bar, 20 μm.

Figure 7.

Immunfluorescence detection of TGase 2 in human eye.
(A) Ciliary body TGase 2 was present in connective tissue
fibers of the ciliary stroma and endothelia and associated
extracellularly with fibers in the stromata of the ciliary body.
(B) In the iris the enzyme appeared to be associated with
endothelia and single cells in the iris stroma. Bar, 20 μm.

Immunofluorescence detection of different TGases in the cynomolgus
monkey eye. (A) Corneal epithelium shows cytoplasmic and
pericellular expression of TGase 1 in the suprabasal layer and in the
uppermost keratocyte layer. The basement membrane and corneal stroma
are negative. (B) Factor XIII was associated with the
intimal layer of arterioles in the conjunctival stroma and
(C) capillaries between bundles of rectus muscle fibers.
(D) Negative control to area in (B). Images were
derived from confocal Z-scans spanning a depth of 10 μm. Bar, 25μ
m.

Figure 8.

Immunofluorescence detection of different TGases in the cynomolgus
monkey eye. (A) Corneal epithelium shows cytoplasmic and
pericellular expression of TGase 1 in the suprabasal layer and in the
uppermost keratocyte layer. The basement membrane and corneal stroma
are negative. (B) Factor XIII was associated with the
intimal layer of arterioles in the conjunctival stroma and
(C) capillaries between bundles of rectus muscle fibers.
(D) Negative control to area in (B). Images were
derived from confocal Z-scans spanning a depth of 10 μm. Bar, 25μ
m.

Visualization of endogenous TGase activity in human ocular tissues
(A) Cornea: TGase activity was present in the corneal
epithelium, its basement membrane, and keratocytes of the superficial
stroma. (B) Conjunctiva: BiotC incorporation was prominent
in the conjunctival epithelium, particularly along the intercellular
spaces. (C) Sclera: BiotC was predominantly incorporated
into episcleral vessel walls. (D) Iris: TGase activity was
present in the dilator muscle, in the walls of stromal blood vessels,
and along delicate fibrillar structures in the stroma, particularly in
the anterior border layer. Bars, 100 μm.

Figure 1.

Visualization of endogenous TGase activity in human ocular tissues
(A) Cornea: TGase activity was present in the corneal
epithelium, its basement membrane, and keratocytes of the superficial
stroma. (B) Conjunctiva: BiotC incorporation was prominent
in the conjunctival epithelium, particularly along the intercellular
spaces. (C) Sclera: BiotC was predominantly incorporated
into episcleral vessel walls. (D) Iris: TGase activity was
present in the dilator muscle, in the walls of stromal blood vessels,
and along delicate fibrillar structures in the stroma, particularly in
the anterior border layer. Bars, 100 μm.

TGase activity in cynomolgus monkey eye. (A) Pars plicata
region of the ciliary body: BiotC was strongly incorporated into the
zonular fibers on the surface and moderately incorporated into the
nonpigmented ciliary epithelium, the connective tissue stroma, and the
ciliary muscle itself. (B) Pars plana region of the ciliary
body: TGase activity was visualized in the zonular fibers on the
surface of the nonreactive ciliary epithelium, in the inner limiting
membrane, the connective tissue layer, the extension of the ciliary
muscle, and the sclera. (C) Retina and choroid: In addition
to the choroidal connective tissue, biotC incorporation was confined to
the walls of the retinal capillaries and, to a minor extent, to the
inner segments of the photoreceptor layer. (D) Retina,
choroid, sclera, and extraocular muscle tissue: TGase activity was
demonstrated in retinal vessels walls, in the choroid and sclera, and
in the vessel walls and margins of individual muscle fibers of
extraocular muscle tissue. (E) Conjunctiva: BiotC was
incorporated into epithelial cells, walls of conjunctival and
episcleral vessels, and fine fibrillar structures in the conjunctival
stroma, particularly concentrating subjacent to the epithelial layer.
(F) Higher magnification of (E). Bars, 100μ
m.

Figure 4.

TGase activity in cynomolgus monkey eye. (A) Pars plicata
region of the ciliary body: BiotC was strongly incorporated into the
zonular fibers on the surface and moderately incorporated into the
nonpigmented ciliary epithelium, the connective tissue stroma, and the
ciliary muscle itself. (B) Pars plana region of the ciliary
body: TGase activity was visualized in the zonular fibers on the
surface of the nonreactive ciliary epithelium, in the inner limiting
membrane, the connective tissue layer, the extension of the ciliary
muscle, and the sclera. (C) Retina and choroid: In addition
to the choroidal connective tissue, biotC incorporation was confined to
the walls of the retinal capillaries and, to a minor extent, to the
inner segments of the photoreceptor layer. (D) Retina,
choroid, sclera, and extraocular muscle tissue: TGase activity was
demonstrated in retinal vessels walls, in the choroid and sclera, and
in the vessel walls and margins of individual muscle fibers of
extraocular muscle tissue. (E) Conjunctiva: BiotC was
incorporated into epithelial cells, walls of conjunctival and
episcleral vessels, and fine fibrillar structures in the conjunctival
stroma, particularly concentrating subjacent to the epithelial layer.
(F) Higher magnification of (E). Bars, 100μ
m.

Immunfluorescence detection of TGase 2 in human eye.
(A) Ciliary body TGase 2 was present in connective tissue
fibers of the ciliary stroma and endothelia and associated
extracellularly with fibers in the stromata of the ciliary body.
(B) In the iris the enzyme appeared to be associated with
endothelia and single cells in the iris stroma. Bar, 20 μm.

Figure 7.

Immunfluorescence detection of TGase 2 in human eye.
(A) Ciliary body TGase 2 was present in connective tissue
fibers of the ciliary stroma and endothelia and associated
extracellularly with fibers in the stromata of the ciliary body.
(B) In the iris the enzyme appeared to be associated with
endothelia and single cells in the iris stroma. Bar, 20 μm.

Immunofluorescence detection of different TGases in the cynomolgus
monkey eye. (A) Corneal epithelium shows cytoplasmic and
pericellular expression of TGase 1 in the suprabasal layer and in the
uppermost keratocyte layer. The basement membrane and corneal stroma
are negative. (B) Factor XIII was associated with the
intimal layer of arterioles in the conjunctival stroma and
(C) capillaries between bundles of rectus muscle fibers.
(D) Negative control to area in (B). Images were
derived from confocal Z-scans spanning a depth of 10 μm. Bar, 25μ
m.

Figure 8.

Immunofluorescence detection of different TGases in the cynomolgus
monkey eye. (A) Corneal epithelium shows cytoplasmic and
pericellular expression of TGase 1 in the suprabasal layer and in the
uppermost keratocyte layer. The basement membrane and corneal stroma
are negative. (B) Factor XIII was associated with the
intimal layer of arterioles in the conjunctival stroma and
(C) capillaries between bundles of rectus muscle fibers.
(D) Negative control to area in (B). Images were
derived from confocal Z-scans spanning a depth of 10 μm. Bar, 25μ
m.